Distribution of multiple antibiotic-resistant Gram-negative bacteria in potable water from hand-dug wells in Iwo, Nigeria

Seventy percent of the Earth's surface consists of water while the remaining is land which contains only 2% potable water (Lim et al. 1999). A major problem facing humanity, especially in an underdeveloped nation, is accessibility to adequate and quality water. With the increase in population in most towns and cities and the corresponding increase in demand for social amenities, it has become very challenging to meet all the water requirements in terms of quantity, quality, and constancy. Nigeria is faced with many challenges in the drinking-water subsector (Akoteyon 2019). The public water supply in Nigeria is mostly non-existent and where available it is inaccessible, the supply is intermittent and unreliable and thus it has become increasingly difficult to meet all the water requirements (Abubakar 2018); this has forced many households to resort to unwholesome water sources that are not potable, resulting in many digging personal boreholes or wells (Balogun et al. 2017).

Apart from the rapid population growth and urbanization, rising demand and falling supplies due to overexploitation and anthropogenic impacts remain some of the major challenges in the public water sector. In addition, low budget and poor investment in water infrastructure, poor policy implementation and lack of political will also contribute to the current low access to safe water supply in the country. The provision of potable water supply and management is one of the vital human needs for healthy living according to the sixth Sustainable Development Goal (SDG), which is geared towards ensuring the availability and sustainable management of water and sanitation for all. Therefore, it is expected that paying adequate attention to urban water supply should be prioritized in urban planning (Akoteyon 2019). The failure of the government in providing safe drinking water led to people sourcing potable water by themselves by digging wells for household use.

Ishaku et al. (2011) noted that the majority of the rural communities in Nigeria lack access to improved water supply. Generally, they rely on free water supply sources such as rivers, perennial streams, ponds and unprotected wells, which are susceptible to water-borne diseases. Pollution of groundwater is one of the major environmental challenges arising from improper and indiscriminate disposal of sewage, industrial and chemical waste.

Findings from several studies are that groundwater is highly contaminated and clinically unsafe for human consumption (Mile et al. 2012). It has been reported that groundwater is easily contaminated by rainstorm overflows, runoff from farming areas and areas with septic systems and latrines that are improperly situated (Sparks 2005).

Pathogens such as Salmonella, Escherichia, Shigella, Vibrio and Campylobacter have been identified in poorly treated water. However, a wide variety of opportunistic pathogens, such as Aeromonas, Pseudomonas and coliforms, are commonly found (Borchardt et al. 2003). Diarrhoeal infections are still a leading child-killer disease worldwide (Walker et al. 2013). Consequently, anyone that consumes such waters is exposed to serious health risks.

Most of the diseases in human beings are caused by unhygienic water supplies used for drinking purposes that cause infections like dysentery, diarrhoea, cholera, typhoid, etc. It has been reported that about 20% of the world's population experiences scarcity of safe drinking water and >5 million people die every year from illnesses associated with drinking water due to inadequate sanitation (Karnwal et al. 2017). It is conceivable that unsafe drinking water contaminated with soil or faeces could act as a carrier of other parasitic infections, such as balantidiasis (Balantidium coli) and certain helminths (species of Fasciola, Fasciolopsis, Echinococcus, Spirometra, Ascaris, Trichuris, Toxocara, Necator, Ancylostoma and Strongyloides and Taenia solium) (Ashbolt 2015). However, in most of these, the normal mode of transmission is ingestion of the eggs in food contaminated with faeces or faecally contaminated soil (in the case of Taenia solium, ingestion of the larval cysticercus stage in uncooked pork) (Ashbolt 2015).

Groundwater may be found almost anywhere on Earth if one digs deep enough, but most accessible groundwater is generally found within 1 km of Earth's surface (Hess 2014). Groundwater is water that exists in the pore spaces and fractures in rocks and sediments beneath the Earth's surface. It originates as rainfall or snow and then moves through the soil and rock into the groundwater system, where it eventually makes its way back to the surface streams, lakes, or oceans (EPA 2022). There are two basic types of groundwater pollution: point sources and non-point sources. Point-source pollution is contamination that can be traced to a particular source such as an industrial site, septic tank, or wastewater treatment plant. Non-point-source pollution occurs diffusely in large areas and includes agricultural, human, forestry, urban, construction, mining, and atmospheric deposition (Sparks 2005).

A great number of different species of bacteria have been isolated from water and many are potential causes of different types of diseases in human beings. The frequent presence of Aeromonas in drinking water raised the question of its role as an enteric pathogen because the production of enterotoxins and/or adhesins had been demonstrated. Bacillus spp. are often detected in drinking-water supplies, even supplies treated and disinfected by acceptable procedures. This is largely due to the resistance of spores to disinfection processes (Bartram et al. 2003). Enterobacter sakazakii is sensitive to disinfectants, and its presence in water can be prevented by adequate treatment (WHO/FAO 2004). Klebsiella spp. are natural inhabitants of many water environments. In drinking-water distribution systems, the organisms can grow and colonize the taps. Klebsiella spp. are also excreted in the faeces of many healthy humans and animals, and they are readily detected in sewage-polluted water (Ainsworth 2004). The presence of Shigella spp. in drinking-water indicates recent human faecal pollution. Control measures that can be applied to manage potential risks include the protection of raw water supplies from human waste, adequate treatment, and protection of water during distribution. Escherichia coli (or, alternatively, thermotolerant coliforms) is a generally a reliable indicator for Shigella spp. in drinking-water supplies (Alamanos et al. 2000).

The presence of the pathogenic V. cholerae O1 and O139 serotypes in drinking water is of major public health importance and can have serious health and economic implications in the affected communities (WHO 2002). Salmonella may be associated with all kinds of food and water. The incidence of typhoid fever decreases when the level of development of a country increases (i.e., controlled water sewage systems, pasteurization of milk and dairy products). Where these hygienic conditions are missing, the probability of faecal contamination of water and food remains high and so does the incidence of typhoid fever (Popoff & Le Minor 2005). P. aeruginosa is predominantly an environmental organism, and fresh surface water is an ideal reservoir. Pseudomonas aeruginosa is the most significant example of bacteria capable of multiplying in water, in contrast to most enterobacteria. This bacterium is frequently isolated from surface water and is also a major concern in mineral water bottling plants, because it is an opportunistic pathogen and can contaminate boreholes and bottling plants (Moreira et al. 1994).

Antibiotic resistance is one of the biggest threats to global health, food security, and development today. Antibiotic resistance can affect anyone, of any age, and in any country. Antibiotic resistance occurs naturally, but misuse of antibiotics in humans and animals is accelerating the process. A growing number of infections – such as pneumonia, tuberculosis, gonorrhoea, and salmonellosis – are becoming harder to treat as the antibiotics used to treat them have become less effective. Antibiotic resistance leads to longer hospital stays, higher medical costs and increased mortality (WHO 2018). Resistance to antimicrobial agents typically occurs by one or more of the following mechanisms: Increased efflux, inactivation of the drug, alteration of the target molecule, and/or reduced cellular uptake. Based on the aforementioned, this study aimed at investigating the distribution and antibiotic resistance profiles of Gram-negative bacteria isolated from potable water samples from hand-dug wells in Iwo, Nigeria. The objectives include the isolation and identification of Gram-negative bacteria from the well water samples as well as comparing the quality of the water samples from covered and uncovered wells and between seasons (late wet and dry seasons, i.e., October and December).

Thirty hand-dug wells were randomly selected and sampled twice within Iwo townships between October and December 2018. One hundred millilitres (100 ml) of water samples were collected from the wells by lowering a sterilized bottle that had been rinsed with the well water sample into the well. Immediately after collection, the samples were labelled and transported in black polyethylene bags within 2 h to the Microbiology Laboratory, Bowen University for analysis. The depth of the well was measured and the water samples collected were analysed for pH using a Hanna handheld pH meter (R102895) (Sule et al. 2014), temperature, and electrical conductivity (Mark et al. 1981).

This was determined by the most probable number (MPN) index method using a 3–3–3 regimen. In this technique, a series of tubes containing MacConkey broth at 10 ml double strength (three tubes), 1 ml single strength (three tubes), and 0.1 ml single strength (three tubes) was used and a positive result was indicated by acid and gas production on incubation at 37 °C for 48 h (Sutton 2010).

One milliliter (1 ml) of each of the well water samples was aseptically transferred into molten sterilized nutrient, cetrimide (Lab M) and MacConkey agar (Lab M) plates using the pour plate technique and incubated accordingly. This was carried out in triplicate. Isolates from primary cultures were aseptically sub-cultured onto fresh media to obtain pure cultures using the streak plate technique. The pure isolates were sub-cultured into already prepared slant bottles for the purpose of identification and characterization which was done using standard and appropriate morphological and biochemical tests as well as using the advanced bacteria identification software (ABIS), Bergey's Manual of Determinative Bacteriology for confirmation (Holt et al. 1994), and Microrao.com.

The antimicrobial susceptibility test for each identified isolate was performed using the disk diffusion method using a multidisc containing eight antibiotics (RapidLabs, UK) (CLSI 2020). Isolates were inoculated into peptone broth and incubated at 37 °C for 16 h. The isolates were standardized to 0.5 McFarland standard and confirmed using a spectrophotometer at 650 nm to give an absorbance reading between 0.08 and 0.13. This will give a value of 10,000,000 cfu/ml of bacteria. The standardized bacteria were seeded onto the surface of freshly prepared, dry-surfaced Mueller Hinton agar using sterile swabs (Adeleke & Owoseni 2020). Using sterile forceps, the antimicrobial discs were placed on the agar plates and incubated at 37 °C for 24 h. All isolates were tested for sensitivity to the following antibiotics: gentamicin (10 μg), ciprofloxacin (5 μg), cefuroxime (30 μg), ceftazidime (30 μg), cefixime (5 μg), ofloxacin (5 μg), augmentin (30 μg) and nitrofurantoin (300 μg). The testing was carried out in duplicate and zones of inhibition were measured using a standard millimetre rule. Values were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) into resistant, intermediate, and sensitive categories (CLSI 2020).

The multiple antibiotic resistance (MAR) index is calculated as the ratio of the number of antibiotics to which an organism is resistant to the total number of antibiotics to which the organism is exposed (Krumperman 1983). The value of MARI is 0.20 and it differentiates the low risk (<0.20) from the high risk (>0.20).

Pathogenicity testing was used to characterize the haemolytic properties of the isolated bacteria. A loopful of each colony was streaked on the surface of sheep blood agar plates and incubated at 37 °C for 24 h and extended for another 24 h when α-haemolysis was observed. Haemolysis was recorded based on colour changes caused by haemolytic zones around the bacterial colonies (Darmawatti et al. 2021).

The data obtained from the study were expressed in absolute values and in percentages. Data obtained were analysed by independent sample t-test and paired sample t-test using SPSS 10.0. Hierarchical cluster analysis of the antibiotic sensitivity and resistance patterns of bacteria isolates was carried out using the word linkage method and squared Euclidean measure.

The depth of the wells varied from 0.61 to 12.4 m depending on the topographical levels. For instance, most of the wells have concrete rims and are covered with painted metal plates and wooden planks while a few were left open. The measurement of water depth from the surface of the ground ranged from 0.61 to 7.32 m (Table 1). There was no significant difference between the mean depth of the covered wells (3.71 ± 0.38) and that of the open wells (2.44 ± 0.56) p > 0.05. The temperature of all the well water sampled was not significantly different for both covered and open wells. The mean temperature of the well water ranged from 25 to 31 °C (Table 1). The pH value of the wells sampled ranged from 4.4 to 10.0. The mean pH value of the covered well (6.39 ± 0.15) was also not significantly different from that of the wells that were left open (6.49 ± 0.29).

The electrical conductivity values of the well water ranged from 20.40–293.00 μs/cm. Statistical results showed that the open wells had a significantly higher value (p < 0.05) than the mean conductivity value of 175.33 ± 35.45 when compared to the mean value for the covered wells (81.41 ± 4.97) as shown in Table 1. Table 2 shows that there was no significant difference between the mean values for pH and conductivity but there was a significant difference in the values for depth and temperature (p < 0.01) at the end of the rainy season and during the dry season (Table 2).

All the 30 wells sampled were found to be grossly contaminated with different bacteria. The results of the MPN fermentation technique showed that the well water samples had very high coliform numbers. The coliform population ranged from 23 to 1,100+ MPN/100 ml for all the wells. Detection of coliform contamination from the well water samples using The MPN technique is shown in Table 3. Twenty-one wells recorded the highest value possible of 1,100+ and it was observed that none was totally free of coliforms.

Eleven genera of Gram-negative bacteria were isolated and identified in this study, namely Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Salmonella, Serratia, and Pseudomonas. Table 4 shows the distribution of the isolated species of bacteria and the number of wells from which the bacteria were isolated. Klebsiella oxytoca was the highest-occurring isolate that was identified from 22 out of 30 wells. This was followed by Proteus mirabilis (from 17 wells), Klebsiella pneumoniae and P. aeruginosa (isolated from 15 wells each) and Pseudomonas sp. (from 13 wells). Two species of Citrobacter were found in only two wells each. Three species of Pseudomonas were isolated from one well each.

The antibiotics susceptibility study of the isolates showed that all the bacterial isolates exhibited resistance to more than three antibiotics, although their pattern of resistance varied. Resistance to cefixime was the highest 92.6%, cefuroxime (90.9%), and ceftazidime (81.7%), all three antibiotics belonging to the cephalosporin class. The other susceptibility patterns are as shown in Table 5.

The MARI of the isolates in this study ranged from 0.13 to 1.00. MARI values greater than 0.2 imply a high level of exposure to antibiotics. Only 6 isolates had MARI values ≤ 0.20 while the remaining 169 isolates had values greater than 0.2 (Table 6).

Table 7 shows the resistance of the bacteria to the different classes of antibiotics as well as the patterns of multiple resistance observed. Klebsiella oxytoca, Citrobacter freundii, and Pseudomonas pseudomallei were found to be resistant to the three cephalosporins tested (ceftazidime, cefuroxime, and cefixime), the fluoroquinolone (ciprofloxacin), and the penicillins (augmentin) drug tested.

Table 8 shows the pathogenic ability of the organisms in producing haemolytic enzymes showing either α or β haemolysis on blood agar. All the isolates exhibited α-haemolysis with the exception of an E. coli isolate which showed β-haemolysis.

Thirty wells were randomly sampled in Iwo and it was observed that all the wells sampled were grossly contaminated with different bacteria genera with counts above the WHO prescribed limit of 0 MPN/100 ml for untreated water (WHO 2002); potable water should be devoid of total coliform in any given sample. A similar result reported total coliform values outside the WHO accepted range (Rogbesan et al. 2002). High MPN values may be a result of the wells constantly receiving polluted water from surface runoff and seepage from contaminated groundwater. Some of the wells are located in very crowded locations receiving doses of faecal materials from the septic tank, water from an abattoir, sewage water, and pit latrines; in addition, some of the wells were uncovered. These results are similar to previous reports of high coliform counts in well and borehole waters analysed (Ngwa & Chrysanthus 2013; Gambo et al. 2015).

The presence of coliform bacteria such as E. coli, Citrobacter, Enterobacter, and Klebsiella species in these well water samples make them unsafe for drinking for human consumption. Members of the coliform group were isolated from stored borehole water (Eniola et al. 2007), in drinking water in rural Peshawar, India (Amin et al. 2012) in well water in Shagamu, Nigeria (Idowu et al. 2015). Water samples were collected from tube well and storage tanks and their results showed that 90% of the samples were positive for coliforms, 40% were faecal coliform positive and 20% were E. coli positive. Karnwal et al. (2017) identified Enterobacter aerogenes and E. coli from drinking water sources. The presence of faecal coliforms is suggestive of the presence of much more dangerous bacteria like Salmonella, pathogenic strains of E. coli, Shigella, etc. (Atobatele & Owoseni 2012). These organisms may bear virulent genes which can pose severe health risks to consumers generally (Biyela et al. 2004).

Pseudomonas sp. has been reported to contaminate some food types and because they produce lipolytic and proteolytic enzymes, the shelf-life quality of the food which they contaminate will be compromised (Raposo et al. 2017). P. aeruginosa is the most significant bacteria that are able to multiply in water, contrary to most enterobacteriaceae (Szita et al. 2007). It is an opportunistic pathogen and can contaminate boreholes and bottling plants. Klebsiella was the highest-occurring genus, the origin of the contamination is not always clear, since Klebsiella species are widely distributed in nature and in the gastrointestinal tracts of a wide range of animals. K. pneumoniae, K. oxytoca, K. variicola, K. terrigena, and K. planticola are commonly found in carbohydrate-rich waste water, surface water, cooling water, soil, plant products, fresh vegetables, sugar cane, frozen orange juice concentrate, and grains. High numbers of K. pneumoniae and K. oxytoca isolates have been isolated from untreated water samples collected from dam, seawater, sediment, and intestinal contents of shrimps and freshwater fishes. The public health significance of Klebsiella in water is an important concern (Gundogan 2014). This is in line with the findings of this study.

Multi-antibiotic-resistant bacteria were present in all (30/30) of the well water samples, and a high number of the identified bacteria (≥80%) were resistant to all antibiotics in the cephalosporins group which is a class of β-lactam antibiotics, namely cefixime, ceftazidime (third-generation cephalosporins), and cefuroxime (a second-generation cephalosporin). The same high level of resistance (83.4%) was recorded against augmentin, a member of the penicillin group which is a combination of amoxicillin and clavulanic acid. Previous studies have observed and reported the identification of multi-antibiotic-resistant bacteria in potable water sources (Su et al. 2018; Ateba et al. 2020) and street-vended foods (Adeleke & Owoseni 2022). The trend tallies with earlier studies that showed resistance towards β-lactam, macrolides, and phenicols (Mulamattathil et al. 2014). In contrast to some findings, four coliform bacteria isolated from different sources were susceptible to ceftazidime (100%) followed by gentamicin and ciprofloxacin with 92% susceptibility (Adeleke & Owoseni 2018). This may serve as a way of noting that environmental samples may be exposed to several antibiotics at a concentration that is more than necessary. The lowest resistance was recorded in ofloxacin (6.3%) followed by gentamicin (18.3%). Most of the multi-antibiotic-resistant bacteria were resistant to the most prescribed classes of drugs, namely, cephalosporins and penicillin. This is a red flag in drug prescription, as these bacteria may develop cross-resistance and this makes treatment of bacterial infections more difficult which may eventually become life threatening.

Multiple antibiotic resistance (MAR) indexing has been shown to be a reliable and cost-effective method of monitoring bacteria sources. MARI is a tool that helps in analysing health risks and checking antibiotic resistance in a given area (Onuoha 2017). In this study, 96.6% of isolates had MARI values greater than 0.2, indicating a high level of exposure of the water in hand-dug wells in Iwo to antibiotics. Previous work has reported the discovery of 91.2 and 66.75% MAR indices of Pseudomonas and Klebsiella species isolates in clinical samples (Osundiya et al. 2013). Production of extracellular enzymes is a microbial virulence factor that helps the microorganism in causing diseases. The ability of the isolated bacteria in this study to produce α or β haemolysis shows a pathogenic side (Darmawatti et al. 2021) in addition to the presence of antibiotic-resistant genes, although not making the isolates pathogenic, it makes transmission of these genes possible which is a great public health issue. It has been reported that haemolysin is an important virulent factor in common E coli infections in the urinary tract and other extraintestinal sites (Bien et al. 2012).

These hand-dug wells which many people use as sources of potable water are contaminated by Gram-negative bacteria that are harbouring several antibiotic-resistant properties as well as the production of extracellular enzyme haemolysin. The presence of the combination of these is harmful and has a great public health significance. This leads to the fact that awareness should be given to the populace on the implication of antibiotic residues in the environment as well as the importance of maintaining a clean and hygienic environment around the wells to ensure the safety of water. It is also advisable that every individual should embark on in-house water treatment in order to avoid water-borne diseases. A recommended distance of 50–100 feet from potential sources of groundwater contamination like soakaways, pit latrines, etc., by health authorities should be maintained.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.